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Most animal-derived major allergens causing respiratory sensitization belong to the family of proteins called lipocalins. Their sequential identity varies but the three-dimensional structure is conserved. They are present in body fluids and secretions. Several lipocalins are able to bind and transport small hydrophobic ligands like retinol. The immunological characteristics of lipocalin allergens are poorly known. Cow dust-derived allergen, Bos d 2, which is a potent inducer of IgE production, was observed to induce the weak proliferative responses of peripheral blood mononuclear cells of asthmatic patients upon stimulation in vitro. The responses were Th2-deviated and directed to a few epitopes in Bos d 2. One of the epitopes, situated adjacent to a structurally conserved region of lipocalins, was recognized by the T cells of all patients. Computer predictions suggested that human endogenous lipocalins may contain epitopes in the corresponding region. We have proposed that the allergenicity of lipocalins may be associated with the adaptation of the immune system to the presence of endogenous lipocalins. [ABSTRACT FROM AUTHOR]

Building on extensive physiological characterization of sodium transport mechanisms along the renal tubule over the past 30 years, complementary DNAs for almost all of the major transporters and channels responsible for renal tubular sodium reabsorption have been cloned over the past 10 years. The consequence is the generation of a broad range of cDNA and antibody probes which can be used to investigate physiological mechanisms on a molecular level. An ensemble of such probes can be exploited for comprehensive analysis of integrative physiological processes, approaches which are referred to as ‘physiological genomics’ or ‘physiological proteomics’. In this review, we describe a targeted proteomic approach to comprehensive analysis of sodium transporter and water channel protein abundance along the renal tubule using an ensemble of rabbit polyclonal antibodies directed to the major sodium transporters and water channels expressed in each renal tubule segment. We discuss preparation and characterization of the antibodies, strategies for quantification of transporter protein abundance, and provide examples of the application of antibody-based targeted proteomics analysis of kidney tissue, revealing the effects of elevations of circulating aldosterone levels and circulating vasopressin levels on sodium transporter, sodium channel, and water channel abundance in kidney. [ABSTRACT FROM AUTHOR]

Cystatins, an amyloid-forming structural superfamily, form highly stable, domain-swapped dimers at physiological protein concentrations. In chicken cystatin, the active monomer is a kinetic trap en route to dimerization, and any changes in solution conditions or mutations that destabilize the folded state shorten the lifetime of the monomeric form. In such circumstances, amyloidogenesis will start from conditions where a domain-swapped dimer is the most prevalent species. Domain swapping occurs by a rearrangement of loop I, generating the new intermonomer interface between strands 2 and 3. The transition state for dimerization has a high level of hydrophobic group exposure, indicating that gross conformational perturbation is required for domain swapping to occur. Dimerization also occurs when chicken cystatin is in its reduced, molten-globule state, implying that the organization of secondary structure in this state mirrors that in the folded state and that domain swapping is not limited to the folded states of proteins. Although the interface between cystatin-fold units is poorly defined for cystatin A, the dimers are the appropriate size to account for the electron-dense regions in amyloid protofilaments. [ABSTRACT FROM AUTHOR]

Many studies have demonstrated a role for ubiquitin (Ub) in the down-regulation of cell surface proteins. In yeast, down-regulation is marked by the internalization of proteins, followed by their delivery to the lumen of the vacuole where both the cytosolic and lumenal domains are degraded. It is generally believed that the regulatory step of this process is internalization from the plasma membrane and that protein delivery to the lysosome or vacuole is by default. By separating the process of internalization from degradation, we demonstrate that incorporation of proteins into intralumenal vesicles represents a distinct sorting step along the endocytic pathway that is controlled by recognition of ubiquitin. We show that attachment of a single ubiquitin can serve as a specific sorting signal for the degradative pathway by redirecting recycling Golgi proteins and resident vacuolar proteins into intralumenal vesicles of the yeast vacuole. This pathway is independent of PtdIns(3,5) P2 and does not rely on the specific composition of transmembrane domain segments. These data provide a physiological basis for how ubiquitination of cell surface proteins guides their degradation and removal from the recycling pathway. [ABSTRACT FROM AUTHOR]

Tenecin 3 is a glycine-rich, antifungal protein of 78 residues isolated from the insect Tenebrio molitor larva. As an initial step towards understanding the antifungal mechanism of tenecin 3, we examined how this protein interacts with the pathogenic fungus Candida albicans to exert its antifungal action. Tenecin 3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles and it did not perturb the membrane potential of C. albicans by the initial interaction. Fluorescence confocal microscopy and flow cytometric analysis revealed that tenecin 3 is rapidly internalized into the cytoplasmic space in energy-dependent and temperature-dependent manners. This internalization is also dependent on the ionic environment and cellular metabolic states. These results suggest that the internalization of tenecin 3 into the cytoplasm of C. albicans is mediated by a fungal cellular process. The internalized tenecin 3 is dispersed in the cytoplasm, and the loss of cell viability occurs after this internalization. [ABSTRACT FROM AUTHOR]

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) have been implicated in membrane-cytoskeletal events underlying cell adhesion, migration, secretion, and phagocytosis. In BV-2 microglial cells, lipopolysaccharide (LPS) elicited a dose-dependent increase in mRNA of both MRP (sixfold) and MARCKS (threefold) with corresponding increases in [[sup 3]H]myristoylated and immunoreactive protein levels. LPS also produced significant increases in protein kinase C (PKC)-β twofold and PKC-ε (1.5-fold). Pro-inflammatory cytokines produced by activated microglia (IL-1β, IL-6, TNF-α) did not mimic LPS effects on MARCKS or MRP expression when added individually or in combination. LPS and IFN-γ produced a synergistic induction of iNOS but not MARCKS or MRP. Induction of MARCKS and MRP by LPS was completely blocked by inhibitors of NF-κB (PDTC) and protein tyrosine kinases (herbimycin A), partially blocked by the p38 kinase inhibitor SB203580, and unaffected by the MEK inhibitor PD98059. LPS induction of iNOS was considerably more sensitive to all these inhibitors. The Src kinase inhibitor PP2 had no effect, while the closely related inhibitor PP1 actually increased LPS induction of MARCKS and MRP. Our results suggest that MARCKS and MRP may play an important role in LPS-activated microglia, but are not part of the neuroinflammatory response produced by cytokines. [ABSTRACT FROM AUTHOR]

An experimental system developed previously for the heterologous expression of c-type cytochromes in Escherichia coli Q1has been adapted to monitor protein transfer across the bacteria's cytoplasmic membrane. Apocytochrome, lacking the haem cofactor and probably in an unfolded state, was readily transferred across the cytoplasmic membrane when fused to a Sec-specific signal peptide. Furthermore, cytochrome fused to a signal peptide regarded as specific for the twin arginine transport (Tat) system was translocated in an unfolded state by the Sec apparatus. After maturation and folding in the cytoplasm, Tat-mediated transfer of holocytochrome to the periplasm occurred. We conclude that, in addition to the nature of the specific signal peptide, the folding state of a particular protein also governs its acceptance by a given transport system. [ABSTRACT FROM AUTHOR]

Many risk factors associated with ischaemic stroke are known, including high levels of fibrinogen or factor VII. Protein Z is a vitamin K-dependent coagulation factor, which was found to promote the assembly of thrombin with phospholipid vesicles that might promote coagulation. Indeed, a low protein Z level may be associated with a varying bleeding tendency. Therefore, we hypothesized that high protein Z levels could induce a hypercoagulable state and performed a case–control study to investigate a potential association between high protein Z plasma levels and ischaemic stroke. We measured protein Z in plasma samples from 157 patients with stroke of unknown aetiology and 192 control subjects. All patients had survived an ischaemic stroke or transient ischaemic attack (TIA) for at least 2 months. We found an increased relative risk of ischaemic stroke with increasing protein Z levels, with an odds ratio of 4·3 [95% confidence interval (CI): 1·7–11] for protein Z plasma levels ≥ 160%. Excluding patients with a history of venous thromboembolism from the analysis, the same result was obtained (odds ratio 4·2; 95% CI: 1·6–11·2). Using a logistic regression model, this association also remained significant (P = 0·04) after adjustment for established risk factors. Our data indicated that a high plasma level of protein Z is an independent risk factor for ischaemic stroke. [ABSTRACT FROM AUTHOR]

In Huntington's Disease (HD), the huntingtin protein (Htt) includes an expanded polyglutamine domain. Since mutant Htt concentrates in the nucleus of affected neurons, we have inquired whether normal Htt (Q16-23) is also able to access the nucleus. We observe that a major pool of normal full-length Htt of HeLa cells is anchored to endosomes and also detect RNase-sensitive nuclear foci which include a 70-kDa N-terminal Htt fragment. Agents which damage DNA trigger caspase-3-dependent cleavage of Htt and dramatically relocate the 70 kDa fragment to the nucleoplasm. Considering that polyglutamine tracts stimulate caspase activation, mutant Htt is therefore poised to enter the nucleus. These considerations help rationalize the nuclear accumulation of Htt which is characteristic of HD and provide a first example of involvement of caspase cleavage in release of membrane-bound proteins which subsequently enter the nucleus. [ABSTRACT FROM AUTHOR]

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro. The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet α-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the ‘lysosome-like’ secretory granules of platelets and melanocytes. [ABSTRACT FROM AUTHOR]

Myosin light chain kinase (MLCK) is a regulatory protein for smooth muscle contraction, which acts by phosphorylating 20-kDa myosin light chain (MLC20) to activate the myosin ATPase activity. Although this mode of action is well-established, there are numerous reports of smooth muscle contraction that is not associated with MLC20 phosphorylation. The kinase activity for the phosphorylation is localized at the central part of MLCK, which is also furnished with actin-binding activity at its N terminal and myosin-binding activity at its C terminal. This article overviews as to how such multifunctional properties of MLCK modify the actin-myosin interaction and presents our observations that the phosphorylation is not obligatory in induction of smooth muscle contraction. [ABSTRACT FROM AUTHOR]

Investigates the formation of disulphide bonds during secretion pathway of proteins through periplasmic-independent type 1 pathway. Formation of disulphide bonds through gram-negative bacteria; Application of single-chain Fv antibody fragment; Interference of premature cytoplasmic oxidation on the secretion process.

Examines the involvement of components in the secretion of proteins that contain twin-arginine leader sequences. Role of Sec-independent Mtt in the transport and targeting of a number of periplasmic proteins; Identification of DmsD as a twin-arginine-binding protein; Significance of eukaryotes and prokaryote in the secretion of proteins.

Reviews the progress made in the identification and functional analysis of components of the lysosome biogenesis machinery. Characteristics of lysosomes; Models for lysosome biogenesis; Role of adaptor protein complexes in lysosomal sorting; Proteins involved in transport to lysosomes; Genetic studies in yeast that provided insight into lysosomal biogenesis; Genes involved in lysosome biogenesis that have no orthologues in yeast.

Chlorophyll (Chl) and total soluble protein decreased and proteolytic activity increased over a 12-day period during dark-induced senescence in detached leaves of Tara, a yellowing cultivar (Y) of Dendranthema grandiflora. In Boaldi, a non-yellowing cultivar (NY), Chl and soluble protein remained near initial levels and little change in proteolytic activity was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins showed no major differences in banding patterns between the two cultivars at day 0; however, all of the resolved proteins were diminished in Tara by day 12. On the other hand, in NY Boaldi, the intensity of the protein bands did not change over the 12-day period. Attached and detached leaves exhibited similar senescence patterns for each cultivar. Ethylene (100 µl l-1 ) accelerated the rate of Chl loss in detached leaves of Tara, but had no effect on Boaldi. These observations suggest that Boaldi is a stay-green genotype, possibly a functional type. The results are discussed in relation to the role of ethylene in chrysanthemum leaf senescence. [ABSTRACT FROM AUTHOR]

Discusses the evolution of abalone sperm lysine. Description of abalone reproduction; Amino-acid sequences of lysine; Description of the crystal structure of the red abalone lysine.

The known functions of the Golgi complex include the sorting, packaging, post-translational modification, and transport of secretory proteins, membrane proteins, and lipids. Other functions still remain elusive to cell biologists. With the goal of identifying novel Golgi proteins, a proteomics project was undertaken to map the major proteins of the organelle using two-dimensional gels, to identify the unknowns using tandem mass spectrometry, and to screen for Golgi residents using GFP-fusion constructs. Multiple unknowns were identified, and the initial characterization of one of these proteins is reported here. GMx33α is a member of a conserved family of cytosolic Golgi-associated proteins with no known homology to any known functional domain or protein. Biochemical analyses show that GMx33α differentially partitions into all phases of multiple detergent extractions, and two-dimensional immunoblots reveal that there are multiple differentially modified forms of GMx33α associated with the Golgi, several of which are phosphorylated. Evidence suggests that these post-translational modifications regulate its association with the Golgi. GMx33α was not found on Golgi budded vesicles, and immuno-electron microscopy co-localizes GMx33α to the trans-face on the same three cisternae as TGN38 in normal rat kidney cells. This work represents the preliminary characterization of a novel family of trans-Golgi-associated proteins. [ABSTRACT FROM AUTHOR]

Tests the hypothesis that interferon (IFN)-gamma and lipopolysaccharide (LPS) regulate the expression of natural resistance associated macrophage protein 2 (Nramp2) and increase the uptake of iron from low relative molecular mass complexes by macrophages. Nitrite determination; Nramp2 mRNA expression by IFN-gamma or LPS.

Saccharomyces cerevisiae possesses three related ammonium transporters, Mep1, Mep2 and Mep3, differing in their kinetic properties and in the level and regulation of their gene expression. The three Mep proteins belong to a family conserved in bacteria, plants and animals, which also includes proteins of the rhesus blood group family. In addition to its role in scavenging extracellular ammonium, the Mep2 protein has been proposed to act as an ammonium sensor, essential to pseudohyphal differentiation in response to ammonium limitation. To pursue the biochemical study of the Mep transporters, we raised polyclonal antibodies against the Cterminal tail of each Mep protein. When electrophoresed on SDS-polyacrylamide gel, the Mep1 and Mep3 proteins migrate as expected from their predicted size, whereas the Mep2 protein migrates as a highmolecular-weight smear. Protein deglycosylation with peptide-N-glycosidase F (PNGase F) indicates that, in contrast to Mep1 and Mep3, Mep2 is an asparagine-linked glycoprotein. Site-directed mutagenesis of the four potential N-glycosylation sites of Mep2 shows that Asn-4 of the protein's N-terminal tail is the only site that binds oligosaccharides. This provides evidence for the extracytosolic location of the Mep2 N-terminus. Consistently, treatment of intact protoplasts with proteinase K leads to specific proteolysis of the N-terminal tail of Mep2. The protein's C-terminus, on the other hand, is protected against protease degradation under these conditions, but digested after protoplast permeabilization, suggesting a cytoplasmic location for this part of the protein. Mep2 glycosylation is not required for pseudohyphal differentiation in response to ammonium starvation, and its absence causes only a slight reduction in the affinity of the transporter for its substrate. [ABSTRACT FROM AUTHOR]

ABSTRACT Non-lethal heat-shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat-shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS-like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non-lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non-lethal ethanol treatment. Pre-treatment with a non-lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non-lethal HS pre-treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance. [ABSTRACT FROM AUTHOR]

Abstract. Many kinds of bioactive peptides which might prevent lifestyle-related diseases are released from food proteins after enzymatic digestion. Inhibitory peptides for angiotensin I-converting enzyme (ACE) having anti-hypertensive effect have been isolated from enzymatic digests of various food proteins. LKPNM, which was isolated from the thermolysin digest of dried bonito was activated 8-fold by ACE itself and showed a prolonged effect after oral administration. Two vasorelaxing peptides, ovokinin and ovokinin(2-7), showing antihypertensive effect after oral administration were obtained from ovalbumin digests. We found that low molecular weight peptides derived from food proteins lowered serum cholesterol without increasing excretion of cholesterol and bile acids. An immunostimulating peptide isolated from an enzymatic digest of soybean protein prevented alopecia induced by cancer chemotherapy. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the ontogenic changes in expression of alternatively spliced forms of NR1 protein that contain the N1 cassette (exon 5) in comparison with the total population of NR1 within the rat cerebellum. The N1 cassette is strongly developmentally regulated in the cerebellum, with >80% of total NR1 protein in the adult rat containing the N1 cassette. In contrast, early in development, <20% of NR1 protein contained this cassette. Rat cortices from the same ages did not show an increase in the percent of NR1 protein expressing the N1 cassette, indicating that the developmental changes in the cerebellum are tissue-specific. As the N1 cassette is known to determine NMDA receptor properties, including spermine sensitivity and decay kinetics of glutamate-induced currents, changes in the characteristics of glutamate-activated currents in granule cells from cerebellar slices were compared at postnatal day 7 versus 14. Glutamate responses exhibited fast deactivation kinetics and reduced potentiation by spermine at day 14 while maintaining sensitivity to an NR2B-selective antagonist. These data are consistent with the possibility that N1 cassette expression may be a factor in the developmental changes in properties of extrasynaptic NMDA receptors in the cerebellum. [ABSTRACT FROM AUTHOR]

Deacclimation of Scots pine (Pinus sylvestris) in northern Finland (65°N) was studied from the perspective of protein metabolism, which was related to nitrogen fertilization. Two-year-old seedlings were fertilized with calcium ammonium nitrate (0, 442 and 884 kg N ha-1) in the summer prior to the study. Needles were harvested for analyses at 3-week intervals during the natural deacclimation period from mid-March to the beginning of June 1998. Deacclimation was followed by monitoring various physiological variables: freezing resistance decreased and osmotic potential increased during the experiment and needle dry weight increased from bud flushing onwards. The concentration of soluble proteins in needles was higher in fertilized seedlings but decreased transiently in the 884 kg N ha-1 fertilized seedlings before budbreak. The abundance of several small polypeptides (17–32 kDa) decreased in the spring. A 60-kDa protein, identified by immunoblotting as a dehydrin, was detected in all treatments. The quantity of this dehydrin decreased with resumption of growth, along with the appearance of 50- and 56-kDa dehydrins. The concentration of these dehydrins decreased during dehardening more rapidly in fertilized seedlings than in the control plants. The fertilized seedlings started to grow earlier than the unfertilized plants. In conclusion, nitrogen fertilization provided good reserves for new growth but did not affect deacclimation of pine needles. [ABSTRACT FROM AUTHOR]

Focuses on a study which presented the structure and function of HFE. Identification of HFE; Structure of HFE; Role of HFE in the pathogenesis of haemochromatosis.

Proper cell function relies on correct protein localization. As a first step in the delivery of extracytoplasmic proteins to their ultimate destinations, the hydrophobic barrier presented by lipid-based membranes must be overcome. In contrast to the well-defined bacterial and eukaryotic protein translocation systems, little is known about how proteins cross the membranes of archaea, the third and most recently described domain of life. In bacteria and eukaryotes, protein translocation occurs at proteinaceous sites comprised of evolutionarily conserved core components acting in concert with other, domain-specific elements. Examination of available archaeal genomes as well as cloning of individual genes from other archaeal strains reveals the presence of homologues to selected elements of the bacterial or eukaryotic translocation machines. Archaeal genomic searches, however, also reveal an apparent absence of other, important components of these two systems. Archaeal translocation may therefore represent a hybrid of the bacterial and eukaryotic models yet may also rely on components or themes particular to this domain of life. Indeed, considering the unique chemical composition of the archaeal membrane as well as the extreme conditions in which archaea thrive, the involvement of archaeal-specific translocation elements could be expected. Thus, understanding archaeal protein translocation could reveal the universal nature of certain features of protein translocation which, in some cases, may not be readily obvious from current comparisons of bacterial and eukaryotic systems. Alternatively, elucidation of archaeal translocation could uncover facets of the translocation process either not yet identified in bacteria or eukaryotes, or which are unique to archaea. In the following, the current status of our understanding of protein translocation in archaea is reviewed. [ABSTRACT FROM AUTHOR]

Abstract In addition to its role in exocytosis, SNAP-25 is essential for axonal outgrowth. In order to identify SNARE proteins involved in neurite growth we have used SNAP-25 antibodies to affinity-purify protein complexes enriched in developing rat brain membrane extracts. We have identified a complex between SNAP-25 and syntaxin 13 predominantly present in brain at embryonic or early postnatal stages. We show that syntaxin 13 is developmentally regulated with a decrease in adult brain. In differentiated neuroendocrine PC12 cells as well as primary cortical neurons the protein is localized to a punctated and tubular staining in the perinuclear region and along processes with high levels in the central region of growth cones. Carboxy-terminally tagged syntaxin 13 was also detected on the plasma membrane by in vivo surface-labelling where it colocalized with SNAP-25. Syntaxin 13 has recently been shown to be implicated in early endosomal trafficking. In our study, colocalization with internalized transferrin in the cell body and along neurites confirmed endosomal location in both compartments. Finally, overexpression of full-length syntaxin 13 enhanced neurite outgrowth in NGF-stimulated PC12 cells, whilst it had no effect on regulated secretion. The data suggest that a syntaxin 13-dependent endocytic trafficking step plays a limiting role in membrane expansion during neuronal development. [ABSTRACT FROM AUTHOR]

The present experiments investigated the trafficking of the vesicular acetylcholine transporter (VAChT) tagged with the enhanced green fluorescent protein (EGFP) in living cholinergic cells (SN56). The EGFP-VAChT chimera was located in endosomal-like compartments in the soma of SN56 cells, and it was also targeted to varicosities of neurites. In contrast, EGFP alone in cells was soluble in the cytoplasm. The C-terminal cytoplasmic tail of VAChT has been implicated in targeting of VAChT to synaptic vesicles; thus, we have examined the role of the C-terminal region in the trafficking to varicosities. A C-terminal fragment tagged with EGFP appeared to be selectively accumulated in varicosities when expressed in SN56 cells. Interestingly, the protein was not freely soluble in the cytosol, and it presented a punctate pattern of expression. However, EGFP-C terminus did not present this peculiar pattern of expression in a nonneuronal cell line (HEK 293). Moreover, the C-terminal region of VAChT did not seem to be essential for VAChT trafficking, as a construct that lacks the C-terminal tail was, similar to EGFP-VAChT, partially targeted to endocytic organelles in the soma and sorted to varicosities. These experiments visualize VAChT for the first time in living cells and suggest that there might be multiple signals that participate in trafficking of VAChT to sites of synaptic vesicle accumulation. [ABSTRACT FROM AUTHOR]

Mitochondria are surrounded by two membranes that contain independent and non-related protein transport machineries. Remarkable progress was recently achieved in elucidating the structure of the outer membrane import channel and in the identification of new components involved in protein traffic across the intermembrane space and the inner membrane. Traditional concepts of protein targeting and sorting had to be revised. Here we briefly summarize the data on the mitochondrial protein import system with particular emphasis on new developments and perspectives. [ABSTRACT FROM AUTHOR]

Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are critical proteins in membrane fusion, in both regulated and constitutive vesicular traffic. In addition, proteins that interact with the SNAREs are thought to regulate fusion. Vesicle-associated membrane protein-2 (VAMP-2) is a SNARE protein involved in insulin-dependent glucose transporter 4 (GLUT4) traffic. VAMP-2 is required for productive GLUT4 incorporation into the plasma membrane. VAMP-associated protein of 33 kDa (VAP-33) is an integral membrane protein that binds VAMPs in vitro, and is hypothesized to be a regulator of VAMPs. In L6 skeletal myoblasts, which display insulin-dependent traffic of GLUT4, we show that VAP-33 colocalized significantly with VAMP-2 using indirect confocal immunofluorescence and biochemical cosegregation. Overexpression of wild-type VAP-33 in L6 myoblasts attenuated the insulin-dependent incorporation of myc-tagged GLUT4 into the plasma membrane, and this response was restored by co-overexpression of VAMP-2 linked to green fluorescent protein. Antibodies to VAP-33 microinjected into 3T3-L1 adipocytes abrogated the insulin-stimulated translocation of GLUT4 to the plasma membrane, as measured in adhered plasma membrane lawns. Immunopurified VAMP-2-containing compartments from L6 myotubes and 3T3-L1 adipocytes showed significant levels of VAP-33. We propose that VAP-33 may be a regulator of VAMP-2 availability for GLUT4 traffic and other vesicle fusion events. [ABSTRACT FROM AUTHOR]

In a healing wound, inflammatory cells undergo apoptosis immediately beneath the leading edge of migrating epithelium. A potential mediator of this apoptosis pattern is p53, a protein with antiproliferative effects. Another protein, bcl-2, is antagonistic to p53 and prevents apoptosis. The purpose of this study was to determine the expression and location of p53 and bcl-2 mRNA and protein in healing wounds of normal and genetically diabetic mice. At various time points, full-thickness skin wounds from nondiabetic and diabetic mice were evaluated for p53 and bcl-2 by immunohistochemistry and in situ hybridization. Apoptosis patterns were also determined using the TUNEL method. Messenger RNA for p53 and bcl-2 were quantitated by competitive reverse transcriptase-polymerase chain reaction. Protein and mRNA for p53 were expressed in the leading edge of migrating epithelium, with apoptosis patterns closely following those of p53 production. p53 mRNA levels decreased soon after wounding, but after a few days, levels increased to greater than baseline. bcl-2 was localized to the wound epithelium, but relative amounts tended to oppose levels of p53, i.e, when p53 increased, bcl-2 decreased and vice versa. Wounds in diabetic animals showed a delayed onset of p53 mRNA expression but had persistently greater levels for longer periods of time. bcl-2 mRNA expression was further delayed in diabetic mice and did not develop to levels as high as p53. Production of both proteins was delayed, consistent with the mRNA expression. Our data show that immediately after wounding, bcl-2 increases and p53 decreases to allow for the cellular proliferation that is required for tissue repair. Over time, bcl-2 levels decrease while p53 levels increase to shut down the inflammatory process and down-regulate the proliferative response. Diabetic animals appear to lose the indirect relationship between p53 and bcl-2. This loss may contribute to the altered apoptosis patterns observed in diabetic healing. [ABSTRACT FROM AUTHOR]

Examines the progress made in characterizing mammalian chromodomain proteins and emphasizes their emerging role in the regulation of gene expression and genome organization. Information on mammalian chromodomain proteins; Role of mammalian chromobox genes in genome organization; Replication of heterochromatin-like complexes; Conclusions.

KIF3 is a heterotrimeric member of the kinesin superfamily of microtubule associated motors. This functionally diverse family of motors is involved in anterograde transport of membrane bound organelles in neurons and melanosomes, mediates transport between the endoplasmic reticulum and the Golgi, and transports protein complexes within cilia and flagella required for their morphogenesis. Interestingly, a mutation of KIF3, which impairs ciliogenesis in nodal cells, prevents the unidirectional leftward flow (nodal flow) of putative morphogens during embryogenesis, thereby altering the development of left–right asymmetry in mammals. [ABSTRACT FROM AUTHOR]

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of two distinct SNARE complexes. Pep12p and Vam3p are both t-SNAREs of the syntaxin family that are components of these SNARE complexes. We have used a genetic approach to address the role of Pep12p in vacuolar protein transport. Our screen for temperature-sensitive pep12 mutants yielded six alleles that were rapidly inactivated upon exposure to the non-permissive temperature. Surprisingly, the proteins encoded by these alleles were all truncated immediately prior to the transmembrane domain. Here we demonstrate that Pep12p requires its transmembrane domain for proper localization, but not for its role in vesicle fusion. In addition, we show that although Pep12p can replace Vam3p in the vacuolar SNARE complex, its transmembrane domain is required to function at this step. Therefore, the transmembrane domain of Pep12p performs different roles in the prevacuolar and vacuolar SNARE complexes. [ABSTRACT FROM AUTHOR]

A number of years ago a small group of investigators was torn apart by a debate over the existence or fate of somewhat obscure endosomal vesicles not even represented in text books. During the last decade, however, interest has shifted with the fireworks of newly discovered molecules involved in the regulation of protein transport. We are now entering the post-genomic era, where individual components can be re-assembled into functional, multiprotein cellular machines. Borders between disciplines in Life Sciences are fading away as our molecular understanding approaches atomic resolution. It is these exciting developments that were captured by the latest edition of the ESF conference series on endocytosis. [ABSTRACT FROM AUTHOR]

Abstract The factors responsible for the induction and maintenance of blood–brain barrier properties are still undefined. The process of blood–brain barrier formation is thought to take place in a two-stage manner: the initial commitment of vascular sprouts by neuroectodermal cells may be followed by the stabilization of barrier properties. In the present study, we investigated the expression pattern of neural (N)-cadherin during early angiogenesis in the brain and the pecten oculi of the chicken. The pecten has been introduced previously as a model for the investigation of the formation and maturation of barrier properties in the central nervous system. Whereas perineural and choroid vessels remained immunonegative for N-cadherin, vascular sprouts invading both the brain and the pecten primordium acquired anti-N-cadherin immunoreactivity. Confocal laser scanning and immunoelectron microscopy indicated that the antigen was located at the ablumenal endothelial membrane in contact with subendothelial cells. With the onset of barrier differentiation as determined by junctional restriction of the tight junction protein occludin, N-cadherin labelling rapidly decreased. Specific intraneuroectodermal upregulation and decline of endothelial N-cadherin was confirmed by in situ hybridization and suggests that N-cadherin expression by cerebral and pecteneal endothelial cells represents an initial and transient signal which may be involved in the commitment of early blood vessels to express blood–brain and blood–retina barrier properties. [ABSTRACT FROM AUTHOR]

Summary: Transport across the nuclear envelope is mediated by transport receptors from the Importin β family. We identified Exportin 1 from Arabidopsis (AtXPO1/AtCRM1) as the nuclear export receptor for proteins carrying leucine-rich nuclear export signals (NESs). AtXPO1 shares 42–50% identity with its functional homologues from humans and yeasts. We functionally characterised AtXPO1 by its interaction with NESs of animal and plant proteins, which is inhibited by the cytotoxin leptomycin B (LMB), and also by its interaction with the small GTPase Ran1 in the yeast two-hybrid system. Furthermore, we demonstrated the existence of a nuclear export pathway for proteins in plants. For the characterisation of nuclear export activities, we established an in vivo assay based on the localisation equilibrium of a GFP reporter protein fused to both a nuclear localisation signal (NLS) and an NES motif. Using this in vivo assay we demonstrated that the NES of the heterologous protein Rev is also functional in plants and that its export is inhibited by LMB. In addition, we identified a leucine-rich NES in the Arabidopsis protein AtRanBP1a. The NES, which is located at the carboxy terminus of the protein, is disrupted by mutating three long chain hydrophobic amino acid residues to alanine (L[sub 176]A, L[sub 179]A, V[sub 181]A). In BY-2 protoplasts the NES of AtRanBP1a is functionally indistinguishable from the Rev NES. Our results demonstrate that the machinery for the nuclear export of proteins is functionally conserved in plants. [ABSTRACT FROM AUTHOR]

Presents information on a study which examined the effect of glycation of bovine beta-lactoglobulin on protein structure. Materials and methods; Modification of the polypeptide chain; Conformational changes; Conclusion.

Summary Eighteen monoclonal Bence–Jones proteins (BJPs) were examined for their effects on cultured LLC‐PK1 (porcine kidney proximal tubule) cells as well as for their amidase and DNase activities. Five proteins were found to enter the cell and to gain access to the nucleus without degradation of epitopes. Intranuclear BJPs ultimately induced DNA fragmentation and cell death. BJPs with relatively high amidase activity were cytotoxic. On the other hand, three of four BJPs with DNase activity had a cytocidal effect on cultured cells; the remaining BJP, which had a relatively high DNase activity but a very low amidase activity, failed to enter the cell and was not cytotoxic in vitro . These results suggest that catalytic and cytotoxic activities of some BJPs may make a significant contribution, in a substantial proportion of myeloma patients, to the development and/or deterioration of the disease. [ABSTRACT FROM AUTHOR]

Comments on the proposal by Albers et al that membrane-anchored secretory proteins in archaea are processed and translocated by a mechanism similar to that of archaeal flagellins. Research findings about archaeal flagellins; Evidence from the author's work on methanogens; Alternative views on the export of proteins by archaea.

Amino acid transport across cellular membranes is mediated by multiple transporters with overlapping specificities. We recently have identified the vertebrate proteins which mediate Na+ -independent exchange of large neutral amino acids corresponding to transport system L. This transporter consists of a novel amino acid permease-related protein (LAT1 or AmAT-L-lc) which for surface expression and function requires formation of disulfide-linked heterodimers with the glycosylated heavy chain of the h4F2/CD98 surface antigen. We show that h4F2hc also associates with other mammalian light chains, e.g. y+ LAT1 from mouse and human which are ∼48% identical with LAT1 and thus belong to the same family of glycoprotein-associated amino acid transporters. The novel heterodimers form exchangers which mediate the cellular efflux of cationic amino acids and the Na+- dependent uptake of large neutral amino acids. These transport characteristics and kinetic and pharmaco- logical fingerprints identify them as y+ L-type transport systems. The mRNA encoding my+ LAT1 is detectable in most adult tissues and expressed at high levels in kidney cortex and intestine. This suggests that the y+ LAT1­4F2hc heterodimer, besides participating in amino acid uptake/secretion in many cell types, is the basolateral amino acid exchanger involved in trans- epithelial reabsorption of cationic amino acids; hence, its defect might be the cause of the human genetic disease lysinuric protein intolerance. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine the time course of GLUT4 protein accumulation following an exercise–carbohydrate supplementation regimen, and to evaluate the effect of this regimen on GLUT4 mRNA regulation. Rats were exercised by swimming and intubated with 1 mL of a 50% glucose solution immediately post-exercise. Exercise significantly reduced muscle glycogen by 50%. By 1.5 h of recovery, muscle glycogen was normalized, but continued to increase above the control level during the next 16 h. A faster and larger repletion of glycogen occurred in the fast-twitch red compared with the fast-twitch white muscle during the 16 h of recovery. GLUT4 protein concentration in fast-twitch red muscle was significantly increased above control by 1.5 h of recovery, and progressively increased throughout the recovery period. Fast-twitch white muscle demonstrated a similar trend, but the increase in GLUT4 protein did not reach significance until 5 h of recovery. Fast-twitch red muscle GLUT4 mRNA was increased by 53% above control immediately post-exercise, but returned to the control level by 1.5 h of recovery. GLUT4 mRNA associated with polysomes, however, increased significantly during this time and remained elevated for a minimum of 5 h. The results suggest that the increased GLUT4 protein expression following a regimen of exercise–carbohydrate supplementation occurs sufficiently fast to contribute to the resynthesis of muscle glycogen, and is controlled by both pre-translational and translational mechanisms. [ABSTRACT FROM AUTHOR]

Focuses on the role of biotin in vitally important pathways in protein, lipid and carbohydrate metabolism in man. Main function of biotinidase; Variation on the development of biotin deficiency of the patients; Indications that central nervous system becomes biotin depleted and more severely than other organs.

Characterizes the translocation machineries which facilitate transport of proteins in mitochondria. Mechanisms of protein sorting into different mitochondrial compartments; Preprotein translocase of the outer cell membrane; Characteristics of target pathways.

Abstract: It has been proposed that mutations in copper/zinc-superoxide dismutase (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), induce the disease by a toxic property that promotes apoptosis. Consistent with this, we have demonstrated that overexpression of Bcl-2, a protein that inhibits apoptosis, attenuates neurodegeneration produced by the familial ALS-linked SOD1 mutant G93A (mSOD1). Herein, we assessed the status of key members of the Bcl-2 family in the spinal cord of transgenic mSOD1 mice at different stages of the disease. In asymptomatic transgenic mSOD1 mice, expression of Bcl-2, Bcl-XL, Bad, and Bax does not differ from that in nontransgenic mice. In contrast, in symptomatic mice, expression of Bcl-2 and Bcl-XL, which inhibit apoptosis, is reduced, whereas expression of Bad and Bax, which stimulate apoptosis, is increased. These alterations are specific to affected brain regions and are caused by the mutant and not by the normal SOD1 enzyme. Relevant to the neuroprotective effects of Bcl-2 in transgenic mSOD1 mice, overexpression of Bcl-2 increases the formation of Bcl-2: Bax heterodimers, which abolish the Bax proapoptotic property. This study demonstrates significant alterations in the expression of key members of the Bcl-2 family associated with mSOD1 deleterious effects. That these changes contribute to the neurodegenerative process in this model of ALS is supported by our observations in double transgenic mSOD1/Bcl-2 mice in which the pernicious increase of Bax is tempered by an increase in formation of Bcl-2: Bax heterodimers. Based on these findings, it may be concluded that Bcl-2 family members appear as invaluable targets for the development of new neuroprotective therapies in ALS. [ABSTRACT FROM AUTHOR]

Abstract: Elucidation of the mechanisms involved in the regeneration of oligodendrocytes and remyelination is a central issue in multiple sclerosis (MS) research. We recently identified a novel alternatively spliced, developmentally regulated oligodendrocyte-specific protein designated microtubule-associated protein-2+13 [microtubule-associated protein-2 expressing exon 13 (MAP-2+13)]. MAP-2+13 is expressed in human fetal oligodendrocytes during process extension and myelination but is minimally expressed in normal mature CNS. To test the hypothesis that MAP-2+13 is reexpressed in regenerating oligodendrocytes in MS lesions, we examined the brains of MS patients for the expression of this protein. By immunocytochemistry using a series of monoclonal antibodies specific for MAP-2+13, we determined that MAP-2+13 expression was up-regulated in all 31 lesions from 10 different MS brains. MAP-2+13 was expressed in regenerating oligodendrocytes associated with demyelinated lesions, with the highest counts found in regions of extensive remyelination. By electron microscopy, MAP-2+13 was localized to oligodendrocytes engaged in remyelination, evident by their process extension and association with thinly myelinated (remyelinated) and demyelinated axons. These results suggest a hitherto unsuspected role for this microtubule-associated protein in oligodendrocyte function during development and myelin repair. [ABSTRACT FROM AUTHOR]

We have previously reported that the FAC1 protein is contained in hippocampal structures that resemble Hirano bodies. Hirano bodies are cytoplasmic inclusions containing actin filaments that are numerous in the hippocampus of many Alzheimer’s disease patients. FAC1 is a developmentally regulated protein that is localized to the cytoplasm of neurons during development and is predominately a nuclear protein in adult brain. In hippocampal sections from non-demented adults, Alzheimer’s disease, and dementia with Lewy bodies patients, Hirano bodies were immunolabelled with antibodies to the FAC1 protein. Confocal laser microscopy demonstrated the presence of actin in FAC1 labelled Hirano bodies, and ultrastructural analysis confirmed the presence of a lattice structure within FAC1 labelled Hirano bodies. Numerous FAC1 immunoreactive swollen dendrites were also present in the hippocampus of Alzheimer’s disease and dementia with Lewy bodies patients. Within any one case the total number of FAC1 positive swollen dendrites correlated with the total number of Hirano bodies, suggesting an association between the two structures. Thus, FAC1 protein is contained in Hirano bodies and swollen dendrites in the hippocampus of patients with Alzheimer’s disease and dementia with Lewy bodies. [ABSTRACT FROM AUTHOR]

Leukocyte cell-derived chemotoxin 2 (LECT2) is a recently isolated protein and has been shown to be synthesized by human hepatocytes. All hepatocytes show diffuse immunostaining for LECT2 within the cytoplasm. In the present study, an attempt was made to clarify the expression pattern of LECT2 in nine cases of low-grade malignant hepatocellular carcinoma (LGM-HCC) and five cases of advanced HCC and 19 cases of premalignant lesion, termed atypical hyperplasia (AH), using the indirect immunoperoxidase technique. Variable spotty to coarsely diffuse staining in the majority of cells, a mixture of positively staining and negatively staining areas, and essentially negative staining was observed within the cellular cytoplasm of AH, LGM-HCC and advanced HCC, respectively. The expression of LECT2 became weaker with the progression of multistep hepatocarcinogenesis. The data clearly demonstrate that LECT2 becomes essentially negative in full-blown HCC cells and that the histological distinction between AH and LGM-HCC is valid. It also seems likely that LECT2 is related to hepatocyte growth. [ABSTRACT FROM AUTHOR]

Studies the physiological role of eosinophil granulocyte in asthma. Consideration of the development of diseases; Ability of eosinophil to produce and secrete potent cytotoxic molecules; Biological activities of proteins.

Focuses on the different adaptive mechanisms used by various organisms especially the molecular mechanism of cold adaptation known as the cold shock response. Discovery of the cold shock mechanism in Escherichia coli; Effects of cold shock on the ribosome function; Role played by the ribosome in cold adaptation.

The effect of constant saturation with water upon Hypogymnia physodes for periods of up to 7 days was investigated in either light or dark, at either 20°C (representative of most laboratory experiments on lichen physiology) or 5°C (more representative of field conditions). In all cases the capacity for photosynthesis, phosphate uptake, and total polyol content declined over 7 days; the decline was greater at 20°C than 5°C, and usually greater in the dark than in the light. In general, both alga and fungus reacted similarly to the treatments. Previously fixed [SUP14]C mostly remained in the polyol pool during the treatments, with relatively little entering the insoluble fraction. The substantial loss of previously fixed [SUP14]C during 7 days saturation was almost entirely from the methanol soluble fraction. Within the insoluble fraction, there was also no loss of [SUP14]C during the 7-day period if protein had been previously labeled with [SUP14]C-leucine. Two other lichens, Xanthoria aureola and Peltigera horizontalis, also showed broadly similar declines to Hypogymnia physodes in various physiological characteristics. [ABSTRACT FROM AUTHOR]